Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders

Definitions

• This invention relates to a novel established cell line.
• This cell line produces a soluble factor which acts on B cells and participates in their differentiation into antibody-producing cells.
• These antibodies are suitable for use in the diagnosis and treatment of rheumatism, autoimmune diseases, immunodeficiency, various infectious diseases, cancer and other diseases in man and animals.
• These antibodies are also useful as antigens for inducing the formation of anti-idiotype antibodies thereto.
• this cell line provides a means very useful, for example, for the analysis of the mechanisms involved in the develoopment of autoimmune diseases, immunodeficiency and the mechanisms involved in the carcinogenesis and metastasis of cancer, and the like.
• mice In order to elucidate the pathology and etiology of autoimmune diseases or the like and make use of the results in the treatment and diagnosis thereof, and in order to analyze the mechanism of immune reactions, an increasing number of studies are being made on animals with autoimmune disease, including mouse strains with autoimmune disease (for example, lupus mouse strains such as NZB mice, NZB/W Fl mice, MRL/MP-lpr/lpr (MRL/t) mice, MRL/Mp-+/+ (MRL/n) mice, BXSB mice are known).
• lupus mouse strains such as NZB mice, NZB/W Fl mice, MRL/MP-lpr/lpr (MRL/t) mice, MRL/Mp-+/+ (MRL/n) mice, BXSB mice are known).
• lymph nodes In MRL/l mice, the lymph nodes begin to swell markedly at the age of about 10 weeks, and abnormal production of autoantibodies (in particular, anti-DNA antibodies ) is observed, and lupus nephritis occurs at high rates.
• autoantibodies in particular, anti-DNA antibodies
• a soluble factor having the effect of inducing the differentiation of B cells is produced by T cells and takes part in the process in which the B cells having been stimulated to divide and grow and differentiate into antibody-producing cells (for example, Dutton, R.W., et al.: Prog. Immunol., 1, 355, 1971; and Schimpl, A., & Wecker, E.: Nature New Biol., 237, 15, 1972).
• TRF T cell replacing factor
• TRF is a humoral factor acting on B cells and it does not induce the proliferation of B cells, but induces the differentiation of B cells into antibody-producing cells. Accordingly, TRF is also called B cell differentiation factor (BCDF).
• BCDF B cell differentiation factor
• BCDF Conventionally known methods for the preparation of BCDF include the method of preparing BCDF by purification from the supernatant of a culture of a T cell hybridoma (Takatsu, K., et al., J. Imunol., 134, 382, 1985) and the method of producing human BCDF by use of cells derived from human B cells (Japanese Patent Laid-Open No. 169424/85).
• BCDF BCDF
• anti-BCDF antibody BCDF
• BCDF can also be utilized for the treatment of immunodeficiency in patients suffering from a reduction in the antibody-producing ability of B cells due to malfunction of helper T cells.
• a useful monoclonal antibody can be efficiently produced by culturing monocloned B cells in vitro and then stimulating their antibody production by the action of BCDF. This antibody can be utilized for the treatment and diagnosis of various diseases.
• BCDF BCDF
• the present inventors have thought that an animal with autoimmune disease will be one producing selectively and abnormally an antibody specific for an autoantigen and the analysis of the pathological state of such an animal will make it possible to elucidate the mechanism involved in the abnormal production of autoantibodies and the develoopment of autoimmune diseases and thereby devise a means useful for the treatment of autoimmune diseases, cancer or the like.
• the swelling of lymph nodes in MRL/l mice is due mainly to the abnormal proliferation of Thyl + , Lytl + T cells
• the present inventors have built up the hypothesis that these T cells of the lymph nodes are producing some factor acting stimulatively on the production of antinucleic acid antibodies or other autoantibodies.
• the KML 1 cell line having self-growing ability has been established by culturing lymph node cells collected from an MRh/l mouse for a long period of time, and not by forming an artificial hybridoma on an extension of the prior art.
• the KML 1 cell line of the present invention produces a factor (TRF or BCDF) participating in the differentiation of antibody-producing cells. It has also been confirmed that this factor is not B cell growth factor (BCGF) because it does not stimulate the DNA synthesis of B cells. It has been one of aims of the present invention to obtain a stable cell line which can be subcultured for a long period of time and which can be subcultured even after freezing and thawing. The present inventors have accomplished this aim by establishing the KMLl cell line having self-growing ability.
• TRF or BCDF B cell growth factor
• the invention provides an established cell line (e.g. KML1), characterised in that it is obtained by (a) collecting lymphoid cells from a mouse or human patient with autoimmune disease, (b) culturing the lymphoid cells in a culture medium containing the supernatant of a culture in which lymphoid cells of animal origin have been grown in the presence of a mitogen, and (c) repeating said step (b) by using a fresh culture medium as defined above.
• KML1 established cell line
• the invention also provides the established cell line ECACC 86 091801.
• the invention also provides a method of producing an established cell line (e.g. KML 1 ) by (a) collecting lymphoid cells from a mouse or human patient with autoimmune disease, (b) culturing the lymphoid cells in a culture medium containing the supernatant of a culture in which lymphoid cells of animal origin have been grown in the presence of a mitogen, and (c) repeating said step (b) by using a fresh culture medium as defined above.
• an established cell line e.g. KML 1
• the KML 1 cell line obtained in the above-described manner consists of cells that can grow stably both when subcultured repeatedly and when frozen, thawed and cultured again. Moreover, the KML 1 cell line has the ability to produce BCDF and, therefore, can be used to produce large amounts of BCDF on an industrial scale. Furthermore, the KML l cell line is also valuable in that it is possible to allow KML 1 cells to act on previously separated antigen-specific B cells and thereby cause the B cells to produce an antigen-specific antibody. In addition, the BCDF produced by the KML 1 cell line is potentially useful because, in some cases, it may act selectively on antigen-specific B cells alone and thereby cause them to produce only antibodies specific for the antigens.
• the frequency of formation of hybridomas producing a desired antibody can be improved by first culturing spleen cells from an immunized animal in the presence of the BCDF obtained with the KML I cell line of the present invention to increase the number of antibody-producing cells, and then subjecting them to cell fusion.
• an antibody specific for the aforesaid BCDF may be produced and used to inhibit the abnormal stimulation of antibody production.
• the KML 1 cell line has self-growing ability, they can be used as parent cells (or partner cells) into which various cells can be fused.
• the formation of useful hybridomas makes it possible to produce a variety of useful physiologically active materials and monoclonal antibodies.
• the KML1 cell line of the present invention provides a means very useful for the diagnosis and treatment of various diseases.
• the invention also provides a method for enhancing the production of anti-nucleic acid antibodies by cells having antibody producing ability characterised (a) by culturing said cells in a culture medium containing the supernatant of a culture of the established cell line of the invention or (b) by culturing cells of the established cell line of the invention and said cells having antibody producing ability in a mixed state.
• the invention also provides the established cell line of the invention for use in enhancing the production of anti-nucleic acid antibodies by an animal by immunizing the animal with a nucleic acid antigen and then administering to the animal the supernatant of a culture of the cell line in combination with nucleic acids.
• the invention also provides B cell differentation factor produced by the established cell line of the invention.
• the invention also provides the established cell line of the invention or B cell differentiation factor according to the invention, for use in diagnosis or therapy.
• the invention also provides the established cell line of the invention for use in the manufacture of B cell differentiation factor for the diagnosis or treatment of rheumatism, autoimmune diseases, immunodeficiency, carcinogenesis or metastatis of cancer.
• the established cell line of the present invention can be obtained by (a) collecting lymphoid cells from a mouse or human patient with autoimmune disease, (b) culturing the lymphoid cells in a culture medium containing the supernatant of a culture in which lymphoid cells of animal origin have been grown in the presence of a mitogen, and (c) repeating the step (b) by using a fresh culture medium as defined above.
• the present invention will be more specifically described below in connection with an embodiment in which an MRL/A mouse is used as the mouse with autoimmune disease and mesenteric lymph node cells are used as the lymphoid cells for the establishment of cell line in the long-term culture of cells.
• a single cell suspension is prepared by collecting mesenteric lymph node cells from a female MRL/£ mouse, aged 20 weeks, and suspending these cells in Dulbecco's modified Eagle medium (DMEM) for the cultivation thereof.
• DMEM Dulbecco's modified Eagle medium
• serum-free media can also be used for the establishment of the cell line and the cultivation of the established cells.
• serum-free media such as Hybrity-1 (manufactured by Sanko Junyaku K.K.) and the like may preferably be used to achieve satisfactory cultivation of the established cells.
• the aforesaid culture medium further contains the following additives.
• interleukin-2 interleukin-2
• the supernatant of a culture in which lymphoid cells of animal origin are grown in the presence of a mitogen is added, as an additive corresponding to IL-2, to the culture medium for the cultivation of mesenteric lymph node cells from an MRL/l mouse.
• This supernatant can be obtained by collecting spleen cells from a female DBA/2 mouse, aged 4-6 weeks; suspending them in DMEM containing the aforesaid additives to form a cell suspension having a cell density of 2 x 10 6 cells/ml; adding thereto 2.5 ug/ml of Concanavalin A (Con A) (an exemplary of mitogen); incubating the cell suspension at 37°C for 24 hours in an incubator having a 5% C0 2 atmosphere; separating the supernatant of the SCS; and then sterilizing the supernatant by filtration through a filter having a pore size of 0.2 p (manufactured by Gelman). This procedure is described in an article by Gills et al. (Gills, S., et al.: Nature, 268, 154, 1977).
• This sterilized filtrate (hereinafter referred to as CAS) is used as an alternative to IL-2. It may be stored in a frozen state at -70°C and thawed just prior to use.
• the established cell line of the present invention can be obtained by using the SCS and CAS prepared in the above-described manner.
• 10% CAS is added to the aforesaid SCS in DMEM containing 5% FCS or in serum-free medium Hybrity-l, and the lymph node cells from MRL/l mice are cultured.
• the cell density in this culture medium should be of the order of 2 x 10 6 cells/ml.
• the culture is carried out, for example, by placing 10 ml of the SCS in a 25 cm 2 flask (No. 25100; manufactured by Corning Glass Works). At intervals of 3 days, all cells are collected by centrifugation at 150xg for 5 minutes and transferred to a fresh culture medium, and no subculture is made until the cells increase in size and come to grow stably. Three months after the start of the culture, the process of transferring the cells to a fresh culture medium at intervals of 3 days is still continued.
• the cells are transferred to a 24-well culture plate (No. 3424; manufactured by Coaster) and cultured at a cell density of 5 x 10 5 cells/ml (2 ml in each well).
• the cells can be cultured in a large-sized flask such as a 75 cm 2 flask (No. 25110; manufactured by Corning Glass Works).
• a large-sized flask such as a 75 cm 2 flask (No. 25110; manufactured by Corning Glass Works).
• the growth of the cells is improved 8 months after the start of the culture.
• the cells acquire self-growing ability and becomes able to grow in the absence of CAS.
• These cells have a doubling time of about 14 hours.
• the established cell line thus obtained will hereinafter be referred to as the KML 1 cell line.
• the KML 1 cell line is a stable one. Its stability has been demonstrated by the fact that, when some cells of the KML 1 cell line being cultured were frozen in liquid nitrogen at -140°C, stored in the frozen state for 3 months, and then returned to the culture system, they grew normally.
• the cell-surface antigens of the KML 1 cell were determined by using antibodies specific for various lymphocyte markers. More specifically, using FITC-labeled antibodies or FITC-labeled biotin-avidin systems (in which the antibody is combined with the biotin and the avidin is labeled with FITC), the cell-surface antigens were detected by laser flow fluoro cytometry (SPECTRUM III; manufactured by Ortho).
• SPECTRUM III laser flow fluoro cytometry
• the KML 1 cell has been found to be Thyl - , Lytl - , Lyt2 and sIg - (surface immunoglobulin-negative), indicating that the KML 1 cell is the so-called null cell.
• H-2 haplotype is k, indicating that the KML 1 cell is derived from the MRL/l mouse.
• the KML 1 cell is Ia-positive.
• the KML 1 cell is Fc receptor-negative (FcR - ).
• FcR - Fc receptor-negative
• BCDF B cell differentiation factor
• the KML cell line of the present invention has the function of producing BCDF, and can be used to prepare a solution containing BCDF (hereinafter referred to as a BCDF solution).
• BCDF solution a solution containing BCDF
• One typical procedure is described below.
• KML 1 cells are suspended in DMEM containing 5% FCS or in serum-free medium Hybrity-1 so as to give a cell density of 5 x 10 6 - 10 7 cells per culture.
• the resulting SCS is incubated at 37°C for 24-48 hours in an incubator having a 5% C0 2 atmosphere.
• the SCS is centrifuged at 2,500 rpm for 5 minutes, and the supernatant is separated and filtered through a 0.2 p sterilizing filter (Acrodisk; manufactured by Gelman Co.) to obtain a BCDF solution.
• a concentrated BCDF solution can be further prepared by concentrating a supernatant separated by centrifuging in the above manner to one tenth of its original volume by using Aquacide III (manufactured by Calbiochem), thoroughly dialyzing the resulting concentrated BCDF solution against DMEM and sterilizing it by filtration.
• Aquacide III manufactured by Calbiochem
• the effect on B cell differentiation of BCDF present in the BCDF solution obtained in the above section a) can be determined according to the following procedure.
• a series of culture media are prepared by adding various amounts of the BCDF solution in DMEM containing 5% FCS and the other additives listed above.
• Spleen cells obtained from a female MRL/ l mouse, aged 4 months, are suspended in each of the culture media so as to give a cell density of 10 6 cells/ml, and then cultured for 4 days.
• the anti-single stranded (ss) DNA antibody titer of the 50% saturated ammonium sulfate fraction of the supernatant of each culture is determined and compared with control.
• the concentration of the antibody protein is determined by taking a 5-ml aliquot of the aforesaid supernatant, adding thereto an equal volume of a saturated ammonium sulfate solution to obtain a 50% saturated ammonium sulfate fraction, centrifuging the mixture at 17,000 rpm for 3 minutes at 2°C on a microfuge (Model 780 Haemofuge; manufactured by Heraeus), and then measuring the absorbance (A 280 ) of the resulting supernatant at a wavelength of 280 nm.
• the activity of the antibody is determined according to the enzyme-linked immunosorbent assay and, basically, the procedure described in Japanese Patent Laid-Open No. 56694/'83, Japanese Patent Application No.
• an ssDNA antigen is prepared by heating dsDNA at 100°C for 5 minutes and then cooling it rapidly.
• a micro titer plate Immulon No. 2; manufactured by Dynatech
• 50 pl each of the aforesaid 50% saturated ammonium sulfate fractions were placed. After the plate is shaken at room temperature for an hour, it is washed three times with Tween/TBS (sodium chloride- containing Tris buffer solution/25 mM Tris hydrochloride + 140 mM sodium chloride, pH 7.4) to remove any unreacted antibodies.
• the activity of the antibody combined with the antigen adsorbed on the plate can be determined by using the activity of the alkaline phosphatase-conjugated second antibody as an index.
• This enzyme activity can be determined, for example, by measuring the absorbance (A 405 ) at a wavelength of 405 nm with a Titertech Multiscan Autoreader (manufactured by Flow Laboratories).
• the BCDF solution obtained by using the KML 1 cell line of the present invention have the effect of stimulating the differentiation of B cells and increasing the number of antibody-producing cells.
• the ability of the BCDF solution to increase the number of antibody-producing cells can be tested, for example, according to the following procedure.
• a series of culture media are prepared by adding various amounts of the BCDF solution obtained in the above section a) to the serum-free medium Hybrity-1.
• these culture media spleen cells obtained from a male MRL/t mouse, aged 5 months, are suspended so as to give a cell density of 10 6 cells/ml, and incubated for 4 days.
• the number of plaque forming cells (PFCs) in each culture is determined. Then, the ability of the BCDF solution to increase the number of antibody-producing cells can be evaluated on the basis of the relationship between the number of PFCs and the amount of BCDF solution added.
• the number of PFCs is obtained by determining the total number of cells producing antibodies including both IgG and IgM.
• PFC assays are made according to the procedure of Gronovicz et al. (Gronovicz, E., et al.: Eur. J. Immunol., 6, 588-590, 1976).
• the number of ssDNA-specific PFCs can be determined according to the procedure of Roder et al. (Roder, J.C., et al.: J. Immunol., 121, 29-37, 1978).
• the BCDF solution obtained with the KML 1 cell line of the present invention has the effect of stimulating in vivo the differentiation of B cells.
• This in vivo effect can be tested, for example, according to the following procedure.
• SRBCs sheep red blood cells
• 4 x 10 8 ssDNA-coated SRBCs are intravenously injected again and, at the same time, 0.2 ml of a BCDF solution is intraperitoneally administered to the mice.
• This BCDF solution is prepared by culturing KML 1 cells in the Hybrity-1 medium for 24 hours, concentrating the supernatant of the resulting culture to one tenth of its original volume by means of Aquacide III, dialyzing the concentrated solution thoroughly against DMEM, and then sterilized it by filtration.
• 0.2 ml of the BCDF solution is intraperitoneally administered to the mice on days 6 and 8.
• spleen cells are collected from the BALB/c mice and the number of PFCs per unit population of spleen cells is determined according to the above-described procedures. As described in Experiment 6 which will be given later, when spleen cells were collected from the BALB/c mice and examined, it was found that the number of IgG PFCs per unit population of spleen cells was approximately doubled. This indicates that the number of antibody-producing cells can actually be increased by in vivo administration of the BCDF solution.
• the presence or absence of a B cell growth factor in the supernatant of a culture of KML 1 cells can be determined as follows.
• a series of single cell suspensions containing spleen cells obtained from a female MRL/R mouse, aged 4 months, are prepared. After the addition of various amounts of the BCDF solution obtained in the above section a), the suspensions are incubated for 3 days. 18 hours before completion of the incubation, 3 H-thymidine is added to the cultures in an amount of 1 uCi/ml. After completion of the incubation, the cells are collected from each culture and tested to determine whether or not the incorporation of 3 H-thymidine was enhanced by the addition of the BCDF solution.
• the KML 1 cell line having the above-described features can be used in a variety of applications.
• One exemplary application utilizing the direct effect of the KML 1 cell line is a method for enhancing the production of anti-DNA antibodies by the mixed cultivation ( M LR) of MRL/l or MRL/n mouse spleen cells with KML 1 cells.
• MRL/n mouse is a mouse which is conjenic with MRL/l mouse, has a deletion of the lpr gene, and does not develop early symptoms of SLE (e.g., swelling of lymph nodes) unlike MRL/l mouse, but exhibits the production of anti-nucleic acid antibodies after a year (see Masanori Shiraki & Michio Fujiwara: Rinsho Men'eki, 15, 15, 1983).
• BCDF produced by the KML 1 cell line of the present invention is thought to be applicable to the treatment of immunodeficiency patients characterized by reduced ability to produce antibodies.
• the present invention has been described in connection with an embodiment in which the MRL/i mouse is used as the mouse with autoimmune mouse and meseteric lymph node cells are used as the lymphoid cells.
• the materials used for obtaining the established cell line of the present invention are not limited thereto. For example, the following materials may also be used.
• lymph node cells include, in addition to meseteric lymph node cells, for example lymph node cells collected from the submandibular lymph nodes, subaxillary lymph nodes, inguinal lymph nodes.
• lymph node cells obtained from MRL/l mice
• lymph node cells for example, obtained from NZB mice, NZB/W Fl mice, BXSB mice, New Zealand mice, SL/Ni mice and the like, as well as peripheral blood lymphocytes, tonsillar lymphoid cells, spleen cells and like cells obtained from human patients with autoimmune disease.
• the supernatant such as CAS
• a culture in which lymphoid cells of animal orgin have been grown in the presence of a mitogen there may used, in addition to spleen cells and lymph node cells obtained from DBA/2 mice, spleen cells and lymph node cells obtained from BALB/c mice, C3H/He mice, C57BL/6 mice, etc.; spleen cells and lymph node cells obtained from Wistar rats, Donryu rats, SD rats, etc.; spleen cells and lymph node cells obtained from guinea pigs, rabbits, dogs, cats, etc.; peripheral blood lymphocytes, spleen cells and lymph node cells obtained from human subjects; and the like.
• a single cell suspension was prepared by obtaining cells of mesenteric lymph nodes from a female MRL/l mouse, aged 20 weeks, and suspending them in DMEM containing 5% FCS and the other additives as described above at a cell density of 2 x 10 6 cells/ml.
• spleen cells were obtained from a female DBA/2 mouse, aged 5 weeks, and suspended in DMEM containing 5% FCS and the other additives as described above to form a cell suspension having a cell density of 2 x 10 6 cells/ml. After the addition of 2.5 ⁇ g/ml of Con A, the cell suspension was incubated at 37°C for 24 hours in an incubator having an 5% C0 2 atmosphere. Then, the supernatant of the resulting culture was sterilized by filtration through a 0.2 Acrodisk filter (manufactured by Nalgen).
• This sterilized filtrate was added to the SCS of the lymph node cells of the MRL/l mouse in an amount of 10%.
• Ten milliliter of the resulting mixture was placed in a 25 cm 2 flask (No. 25110; manufactured by Corning Glass Works) and the culture was started. At intervals of 3 days, all cells were collected by centrifugation at 150xg for 5 minutes and transferred to a fresh culture medium, and no subculture was made until the cells increased in size and came to grow stably. Three months after the start of the culture, all the cells increased in size, but still continued to grow slowly. Thus, the process of transferring the cells to a fresh culture medium at intervals of 3 days was continued.
• the cells were transferred to a 24-well culture plate (No. 3424; manufactured by Coaster) and cultured at a cell density of 5 x 10 5 cells/ml (2 ml in each well). At this stage, the growth of the cells was improved and, thereafter, the cells became to grow in a 75 cm 2 large-sized flask (No. 25110; manufactured by Corning Glass Works). At this point of time, or 8 months after the start of the culture, these cells became able to grow in the absence of CAS and were judged to have acquired self-growing ability.
• the cell line thus obtained was named K ML 1 .
• KML 1 KANAI MRL-One-Seven
• ECACC European Collection of Animal Cell Cultures
• KML 1 cell were frozen in liquid nitrogen at -140°C and stored in the frozen state. After a month, the cells were thawed and cultured again. These cells grew stably and exhibited the same properties as those being subcultured.
• KML 1 cells were injected into the abdominal cavity of female nude mice (BALB/c-nu/nu). After 2 months, the formation of lymphomas was observed. Thus, it has been confirmed that KML 1 cells can take as tumor cells in nude mice.
• KML 1 cells were suspended in DMEM containing 5% FCS and the other additives as described above in an amount of 5 x 10 6 -10 7 cells per culture, and grown at 37°C for 48 hours in an incubator having a 5% C0 2 atmosphere. Immediately after that, the resulting culture was centrifuged at 2,500 rpm for 5 minutes. The supernatant was separated and sterilized by filtration through a 0.2 p Acrodisk filter to obtain a BCDF solution.
• Example 4 The procedure of Example 4 was repeated except that the DMEM was replaced by the serum-free medium Hybrity-1 and the KKL 1 cells were grown for 24 hours instead of 48 hours. The supernatant of the resulting culture was separated and sterilized by filtration.
• Example 5 Using Aquacide III, the supernatant obtained in Example 5 was concentrated to one tenth of its original volume. The resulting concentrated BCDF solution was thoroughly dialyzed against DMEM and then sterilized by filtration in the same manner as described in Example 4.
• the cell-surface antigens of the KML 1 cell were detected by laser flow fluorocytometry (SPECITRUM III; manufactured by Ortho).
• SPECITRUM III laser flow fluorocytometry
• a series of culture media were prepared by .adding various amounts. of the BCDF solution obtained in Example 4 to DMEM containing 5% FCS and the other additives as described above.
• spleen cells obtained from female MRL/l mice, aged 4 months, were suspended so as to give a cell density of 10 6 cells/ml.
• These suspensions were placed in 25 cm 2 flasks (No. 25110; manufactured by Corning Glass Works) and incubated for 4 days. Then, the anti-single stranded (ss) DNA antibody titer (A 405 ) of the supernatant of the resulting cultures was determined.
• the antibody titer was expressed in terms of activity per unit amount of antibody protein (1A 280 ).
• this BCDF solution had the ability to enhance the production of anti-DNA antibodies by a factor of about 2.5.
• Spleen cells obtained from female MRL/l mice, aged 4 months, and KML 1 cells were mixed in a culture medium and cultured at 37°C for 2 days in an incubator having a 5% C0 2 atmosphere.
• the spleen cells were suspended at a cell density of 10 6 cells/ml, while the KML 1 cells were suspended at each of the various cell densities shown in the following table.
• the antibody titer was expressed in the same manner as described in Experiment 2.
• Spleen cells obtained from female MRL/n mice, aged 9 months, and KML 1 cells were mixed in a culture medium and cultured under the same conditions as described in Experiment 3.
• the spleen cells were suspended at a cell density of 10 6 cells/ml, while the KML 1 cells were suspended at each of the various cell densities shown in the following table.
• the antibody titer was expressed in the same manner as described in Experiment 2.
• a series of culture media were prepared by adding the BCDF solution obtained in Example 5 to the serum-free medium Hybrity-1 in the various amounts shown in the following table.
• spleen cells obtained from male MRL/l mice, aged 5 months, were suspended so as to give a cell density of 10 6 cells/ml and cultured for 4 days under the same conditions as described in Experiment 2.
• the number of plaque forming cells was obtained by determining the total number of cells producing antibodies including both IgG and IgM according to the above-described procedure of Gronovicz et al.
• the number of ssDNA-specific PFCs was determined according to the procedure of Roder et al.
• SRBCs sheep red blood cells
• spleen cells were collected from the BALB/c mice and the numbers of ssDNA-specific IgG(y) PFCs and IgM(p) PFCs per unit population of spleen cells were determined. Four-measurements were made for each mouse.
• the number of yPFCs in the mice treated with the BCDF solution was approximately doubled as compared with untreated mice. This clearly indicates that the number of antibody-producing cells can also be actually increased by in vivo administration of a BCDF solution obtained with the KML 1 cell line.

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• 1985
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